Dissection of Zebrafish Craniofacial Tissues Upon Staining with Alcian Blue

Summary

The protocol described here presents a simple and easy-to-follow procedure for staining and dissecting craniofacial cartilages in a 5-day-old zebrafish larva. It can be used to study the anatomy, shape, and size of these structures under various developmental conditions.

Abstract

Zebrafish, given its optical transparency, is an excellent vertebrate model to study the mechanisms by which craniofacial cartilages form in the embryo. Craniofacial cartilages can be broadly classified into two groups, the neurocranium and the viscerocranium, which in turn can be subdivided into numerous cartilage groups necessary for supporting the brain and constituting the feeding and respiratory apparatus, among others. This protocol will first describe a simple and established staining procedure in zebrafish to visualize these cartilage groups, followed by a methodology to dissect and separate the neuro and the viscerocranium. From these dissections, simple shape and size metrics can be easily obtained, and in this protocol, this is demonstrated for the palate, which is part of the anterior neurocranium, a tissue often affected in most craniofacial disorders in humans. The protocol can be replicated in a straightforward manner even in resource-limited settings, thus providing a valuable educational tool for undergraduate students to get introduced to craniofacial morphology.

Introduction

The craniofacial cartilage in vertebrates emerges from a multipotent cell population called the cranial neural crest cells¹. These cells are first specified in the dorsal margins of the neural plate in early embryos, which then migrate large distances to reach the craniofacial region and the pharyngeal arches before differentiating^2,3. The signaling mechanisms that drive this migration and differentiation are largely conserved among vertebrates^4,5. Despite this, the organization, size, and shape of individual cartilage elements ....

Protocol

The zebrafish maintenance and experimental procedures used in this study were approved by the institutional animal ethics committee, vide Reference TIFR/IAEC/2023-1.

1. Reagent preparation

1. E3 media:
   1. Prepare 50x stock 1 by mixing 3.25 g of sodium phosphate dibasic (0.458 mM Na₂HPO₄), 0.29 g of potassium dihydrogen phosphate (0.042 mM KH₂PO₄), 11.93 g of sodium chloride (4.084 mM NaCl), and 0.48 g of potassium chloride (0.128 mM KCl) in 1 L of distilled water. 
   2. Prepare 50x stock 2 by mixing 2.43 g of calcium chloride (0.33 mM CaCl₂)....

Results

The zebrafish craniofacial skeleton is fully cartilaginous by 5 days post fertilization (dpf) and thus, can be stained using Alcian blue, which is a basic dye capable of binding to glycosaminoglycans present abundantly in cartilages²³. This results in the cartilage being stained blue (Figure 1) so that it can be distinguished from other tissues present in the larva using a standard stereo microscope available in most undergraduate lab settings. Once the yolk is scrape.......

Discussion

Craniofacial malformations constitute a majority of all birth defects in humans, often leading to infant mortality. However, the underlying etiology in most craniofacial anomalies remains to be elucidated²⁵. Craniofacial structures are complex in any given vertebrate, and from an undergraduate setting perspective, the first requisite is therefore to have an educational tool that provides students a general overview of distinct structures in the craniofacial region. However, having access to a mamm.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agarose	Lonza bioscience	50004	For coating petri dishes
Alcian blue 8G	Sigma-Aldrich	A3157	For staining cartilages
Calcium Chloride dihydrate	Sigma-Aldrich	12022	For making buffers
Camera	Olympus	DP28	For acquisition of images
DAPI	Roche Life Sciences	10236276001	For labelling nuclei
Ethanol	Honeywell	32221	Solvent for alcian blue stain
FIJI			Version: ImageJ 1.54f
Forceps	Dumont	Dumont	For performing dissections
Glycerol	MP biomedicals	193996	For making washes and mounting samples
Hydrogen peroxide	MP biomedicals	194057	For making bleach solution
Magnesium chloride hexahydrate	SARD Biosciences	SBRC0217	For making buffers
Magnesium sulfate heptahydrate	Sigma-Aldrich	M2773	For making buffers
MS-222 (Tricaine)	Sigma-Aldrich	A5040	For anaesthetising larvae
Potassium Chloride	Sigma-Aldrich	P9541	For making buffers
Potassium hydroxide	Sigma-Aldrich	484016	For making buffers
Potassium phosphate monobasic	Sigma-Aldrich	P0662	For making buffers
Sodium Chloride	Sigma-Aldrich	S3014	For making buffers
Sodium phosphate dibasic	Sigma-Aldrich	71640	For making buffers
Stereoscope	Olympus	SZX61	For performing dissections
Tween-20	HIMEDIA	TC287	Detergent, used for permeabilising cells